Preliminary Human Study to Determine the
Bioavailability of
The Right C® and Another
Vitamin C
In a Blinded Cross-over Study
This study was conducted at Kilden
Helse in Oslo, Norway under the direction of Dr. Roald Strand. The
protocol was designed by Dr. Anthony J. Verlangieri, Professor of
Pharmacology and Toxicology.
Purpose
To determine the rate of oral
absorption of The Right C®
and Another Vitamin C by analysis of Total Vitamin C (ascorbic acid,
AA) delivered to plasma at 90 minutes post-ingestion.
Study Design
Ten (10) healthy male subjects were
randomized into two groups as described below. They were to refrain
from taking any vitamin supplements for at least 7 days prior to the
start of the study. They were to avoid intake of fruits and vegetables
prior to and during the study period. The study required that all
subjects fast prior to the administration of the test material.
The study groupings consisted of two groups. Prior to the cross-over,
half of the subjects received 1000 mg of Another Vitamin C orally, and
the other half received 1000 mg of The Right C®.
After a 7 day wash out period, the cross-over treatments began, with
the groupings being reversed.
Prior to administration of the test material, a blood sample was
collected in a heparin tube (0 time, baseline). Blood samples were
then taken at 30 min., 60 min., and 90 min. after administration of
the test material.
Plasma AA was analyzed by HPLC as described below. All results are
expressed as mg AA/L of plasma.
Materials
Ascorbic acid, dithiothreitol (DTT),
tetrasodium EDTA, chloroacetic acid, sodium hydroxide (NaOH), octyl
sodium sulfate, and meta-phosphoric acid (MPA) were purchased from
Sigma-Aldrich Chemical Co., Milwaukee, WI. High Pressure Liquid
Chromatography (HPLC) instrumentation platform included an Alliance
2695 Separations Module (Waters Inc., Milford, MA) with an autosampler,
a Waters 2996 Photodiode Array Detector utilizing Empower Pro
Software. The column used was a 250 x 4.6 Ultrasphere column (Beckman
Instruments, Inc., Fullerton CA) packed with 5-µm octadecylsilane
particles. All solvents were degassed with nitrogen prior to use.
Methods
Isocratic elution at a flow rate of
0.5 mL/min at ambient temperature was used, and the mobile phase
consisted of a solution of 14.1 g chloroacetic acid, 4.65 g NaOH, 0.85
g of tetrasodium EDTA, and 200 mg of octyl sodium sulfate in 1L of
distilled water. The photodiode array detector was set at a wavelength
of 265 nm. Standard curve measurements produced a standard curve with
R=0.9931. Ascorbic acid had a retention time of 6.1 min +/- 0.2 min.
Stock solutions of MPA (30 g/L) and DTT (1 g/L) were prepared and
stored cold. Ascorbic acid standards were prepared and analyzed as
follows. Ascorbic acid (2.0 mg) was weighed and added to a solution
containing 0.7 mL of MPA solution (30 g/L) and 0.3 mL of DTT solution
(1 g/L). This 2.0 mg/mL stock solution was serial diluted to generate
final ascorbic acid standards ranging from 1.25 µg/mL to 20 µg/mL.
Human blood samples were collected (3.0 mL), centrifuged, and the
plasma (0.5 mL) was supplemented with 0.5 mL of MPA (30 g/L) solution
for stability, and frozen at -78°C. After the plasma samples were
received (in duplicate), each were treated with 0.2 mL of stock MPA
solution, and 0.02 mL of stock DTT solution, vortexed, and centrifuged
at 2000xG. Final concentrations of MPA and DTT were 17.2 g/L and 0.2
g/L, respectively. The supernatant (1.0 mL) was transferred to a
microfuge tube, stored on ice, and further centrifuged at 13200xG for
1 minute to remove residual suspended particles. The clear supernatant
plasma solution was immediately transferred to autosampling vials and
analyzed for ascorbic acid. Injection volumes used for standards and
plasma samples was 10 µL. A dilution factor of 2.24 was used for
measuring final ascorbic acid concentrations based on the method
described above. Plasma ascorbic acid concentrations were reported in
mg/L.
Results
The data clearly shows that The Right
C® has significantly
better absorption compared to Another Vitamin C. Figure 1
displays the total plasma AA concentration, correcting for baseline
values, over time. A Two-Way Repeated Measures Analysis of Variance
was performed. The results show that both the main effects of "Time"
and "Type of C" were significant (p=0.0001 and p=0.0002,
respectively). There was not a significant interaction effect.
Table 1 depicts the mean value for Another Vitamin C and The
Right C® at each time
point.
Table 1 Average increase, above baseline, in total plasma AA (mg/L) at each
time interval after ingestion of the test material
Time
(Min)
Another
Vitamin C
The Right C®
30
1.322
4.295
60
4.170
8.331
90
6.222
11.61
Following the Two-Way Repeated
Measures Analysis of Variance, t-test post-hoc analysis was performed
comparing Another Vitamin C and The Right C®
at each time interval.
Figure 2 shows the comparison
between Another Vitamin C and The Right C®
for increase in total plasma AA (mg/L) above baseline at 30 minutes.
T-test post-hoc analysis shows that The Right C®
had a significantly higher increase in total plasma AA above baseline
compared to Another Vitamin C (p=0.0188).
Figure 3 shows the comparison
between Another Vitamin C and The Right C®
for increase in total plasma AA (mg/L) above baseline at 60 minutes.
T-test post-hoc analysis shows that The Right C®
had a significantly higher increase in total plasma AA above baseline
compared to Another Vitamin C (p=0.0278).
Figure 4 shows the comparison
between Another Vitamin C and The Right C®
for increase in total plasma AA (mg/L) above baseline at 90 minutes.
T-test post-hoc analysis shows that The Right C®
had a significantly higher increase in total plasma AA above baseline
compared to Another Vitamin C (p=0.0100).
Absorption rate was calculated and is
graphically depicted in Figure 5. It was assumed that
absorption would be linear over time, and linear regression supports
this assumption (R2=0.50575). The absorption rate of The Right C®
was 196% higher than the absorption rate of Another Vitamin C. Put
another way, the absorption rate of Another Vitamin C was only 50.7%
that of The Right C®.
Assuming that the absorption rate of either source of ascorbic acid
would continue to be linear over time, and that total absorption would
be complete after 4 hours, the total amount of ascorbic acid delivered
to the plasma from a 1000 mg dose of The Right C®
would be 31.883 mg/L, compared to a 16.187 mg/L increase after a 1000
mg dose of Another Vitamin C. Calculating the area under the curve (AUC)
of The Right C® and
Another Vitamin C shows that the AUC of The Right C®
is 205% higher than that of Another Vitamin C (488.6 units versus
238.3 units, respectively).
Discussion
The data indicates that The Right C®
formulation is absorbed more rapidly than Another Vitamin C by 196%.
Alternatively, Another Vitamin C is absorbed only 50.7% as quickly as
The Right C®. The Right
C® raises plasma AA
levels more rapidly and higher than Another Vitamin C. Higher plasma
levels promote more rapid increases in intracellular AA levels. Higher
AA levels enable the cell to utilize AA at a higher rate in cell
metabolism and provide superior anti-oxidant action in the plasma as
well as in the cell. Thus, The Right C®
provides superior anti-oxidant protection compared to Another Vitamin
C.
The Right C® is a registered trademark of The C Group, Inc.
